1. Field
This invention relates generally to immobilized, biologically active, nitrogen-containing, organic substances such as antigens, antibodies, antibiotics, enzymes and other proteins. The immobilized substances comprise insoluble carrier materials on which the said biologically active substances are bound. The binding may be by absorption, adsorption, chelation or other chemical coupling and the insoluble carrier materials may be organic or inorganic, metallic or non-metallic, porous or non-porous. As used herein the terms "immobile", "immobilized" and "immobilization", when applied to biologically active substances, refer to such biologically substances which have been rendered essentially insoluble by binding them to supporting carrier material in such a way that the resulting composite materials retain the original biological activity of the bound substances (such as enzymatic, coenzymatic, antibody or antigenic activity). More particularly, the present invention relates to the preparation of immobilized, biologically active, nitrogen-containing, organic substances (such as enzymic proteins) using supporting carrier materials formed by novel methods with the result that the biological activity of the immobilized biologically active substances is enhanced and displays a prolonged lifetime. According to the present invention, before attachment of the biologically active substance, the carrier material is treated by a process which causes a hydrous metal oxide to be deposited thereon; the metal-oxide-coated carrier material then is used as the support upon which the biologically active material is immobilized. Such carrier materials may be non-porous in which case the metal oxides are coated on their outer surfaces, or they may be porous in which case the metal oxide can be coated on the surfaces within the pores as well as on the outer surfaces.
2. Prior Art
Various methods of attachment of enzymic proteins to organic and inorganic insoluble carriers have been disclosed in U.S. Pat. No. 3,928,143. U.S. Pat. Nos. 3,556,945 and 3,519,538 respectively disclose immobilization of enzymic proteins on inorganic supports by adsorption and by chemical binding using silane coupling agents.
U.S. Pat. No. 3,783,101 discloses a more-water durable immobilized enzyme which comprises a thin, continuous layer of a metal oxide on the surface of a porous glass carrier, with the enzyme bonded to the metal-oxide layer through a silane coupling agent; the method of applying this coating as disclosed in the last mentioned patent amounts to merely contacting the porous glass with a metal solution followed by heating; in each such example shown in U.S. Pat. No. 3,783,101 the resulting initial immobilized enzymic activity in the cases of the metal-oxide-coated carriers or supports was less than, or about equal to, the activity of corresponding immobilized enzymes on the same carriers or supports which did not have the applied metal-oxide coating. The present invention discloses how to make an improved, immobilized biologically active substance (such as an enzyme) supported on metal-oxide-coated carriers in which the metal-oxide coating is applied by a novel method leading to immobilized substances which are unique to the method of coating the support with hydrous metal oxide, and which often show markedly enhanced specific biological activity (e.g. catalytic activity in the case of enzymes) as compared with the corresponding immobilized biological substances attached to the same carrier support material which has not been treated with a coating of metal oxide by our novel and unique method.
U.S. Pat. No. 3,841,969 discloses a process of preparing immobilized enzymes using supporting carriers prepared by reaction of the carrier material with an aqueous solution of a metal salt, drying and washing the resulting solid material, and then reacting this material with an enzyme so as to chelate the enzyme to the metal complex on the supporting material. In the present invention however a coating of metal oxide is formed by hydrolysis of a metal-salt solution; the metal oxide so formed is caused to deposit on the surface of the carrier material thereby conferring on the carrier material improved properties for immobilizing biologically active, nitrogen containing, organic substances such as enzymes and other proteins, by adsorption and by other immobilization techniques known in the art. The novel technique of the present invention differs from that of U.S. Pat. No. 3,841,969 in that our invention teaches the deposition on the carrier of an insoluble metal oxide formed according to the methods disclosed below.
U.S. Pat. No. 3,912,593 discloses water-insoluble, metal complexes of biologically active, nitrogen-containing, organic substances in which these complexes are formed by mixing the biologically active nitrogen-containing organic substances with a hydrous metal oxide capable of complexing with said substance. The present invention teaches how to prepare related substances by a novel process in which the metal oxide which complexes with said biologically active substance is coated by a novel method on a carrier material before contacting with the biologically active material.
The metal oxides employed in the present invention are those insoluble in aqueous solutions of about the pH whereat the respective enzyme displays maximum activity. Ordinarily this will be within a pH range of about 3 to 11. Any metal salt can accordingly be employed which will react to precipitate an oxide when the pH of its solution is adjusted to between about 3 to 11. Metals which are desirable in this regard are Ti, Zr, Sn, Fe, Al, V, Hf with Ti, Al, Sn and Zr preferred. Enzymes which can be immobilized according to the present invention include amyloglucosidase, lactase, glucose oxidase, invertase, trypsin, glucose isomerase, catalase, pronase, urease, lactate dehydrogenase, amino acid acylase, penicillin acylase, proteases and dextranase. Other biologically active substances which can be immobilized according to the present invention include antibodies, coenzymes such as nicotinamide adenine dinucleotide, antibiotics such as penicillin, gramicidin, lathumycin, neomycin, polymyxin, streptomycin, ampicillin and chloroamphenicol, and whole cells such as those of bakers' yeast and Escherichia coli.